Fiber-optic physiological probes

ABSTRACT

Fiber optic sensors suitable for monitoring physiological analyte concentrations are described. The sensors include analyte permeable matrices disposed in a light path defined by the axial core at one end of an optical fiber segment. The matrix contains an indicator molecule covalently linked to a polymer or admixed with the polymer. The indicator molecule may be an absorptive molecule or a luminescent molecule. pH, PCO 2 , and PO 2  sensors are described and may be disposed together in substantially coterminal arrangement to make a multivariable probe.

This is a continuation of the prior application Ser. No. 07/491,336,filed Mar. 9, 1990, U.S. Pat. No. 5,000,901 which in turn is adivisional of application Ser. No. 07/224,620, filed on Jul. 25, 1988,now U.S. Pat. No. 4,925,268. The benefit of the filing dates of whichare hereby claimed under 35 U.S.C. § 120.

FIELD OF THE INVENTION

This invention relates to fiber-optic sensors suitable for monitoringphysiological pH and blood gas concentrations.

BACKGROUND OF THE INVENTION

In recent years fiber-optic chemical sensors have been developed todetect the presence and monitor the concentration of various analytes,including oxygen, carbon dioxide, glucose, inorganic ions, and hydrogenion, in liquids and in gases. Such sensors are based on the recognizedphenomenon that the absorbance and in some cases the luminescence ofcertain indicator molecules is specifically perturbed in the presence ofcertain analyte molecules. The perturbation of the luminescence and/orabsorbance profile can be detected by monitoring radiation that isabsorbed, reflected, or emitted by the indicator molecule whenilluminated in the presence of a specific analyte. Fiber-optic probeshave been developed that position an analyte-sensitive indicatormolecule in a light path that is typically made up of a pair of opticalfibers. One fiber transmits electromagnetic radiation from a lightsource to the indicator molecule; the other fiber transmits the returnlight from the indicator molecule to a light sensor for measurement. Theindicator molecule is typically housed in a sealed chamber whose wallsare permeable to the analyte.

For example, the fiber-optic pH probe disclosed in U.S. Pat. No.4,200,110 includes an ion-permeable membrane envelope which encloses thedistal ends of a pair of optical fibers. The envelope is a short sectionof dialysis tubing which fits closely about the two fibers. ApH-indicating dye-containing solid material, e.g., phenol red/methylmethacrylate copolymer, is packed tightly within the membrane distal tothe ends of the fibers. Cement is applied to seal the distal end of themembrane and also the proximal end where the optical fibers enter themembrane. The membrane has pores of a size large enough to allow passageof hydrogen ions while being sufficiently small so as to precludepassage of the dye-containing solid material. The probe operates on theconcept of optically detecting the change in color of the pH-sensitivedye, e.g., by monitoring the green (570 nm) intensity of phenol red. Oneof the fibers is connected at its proximal end to a light source, whilethe other fiber is connected at its proximal end to a light sensor.Light is backscattered through the dye from one fiber into the otherfiber. In preparing the dye-containing material, light scatteringpolystyrene microspheres of about 1 micron diameter may be added priorto incorporation of the dye material into the hollow membrane. Asimilarly constructed fiber-optic oxygen probe, employing a fluorescentdye sensitive to oxygen quenching, is disclosed in U.S. Pat. No.4,476,870.

U.S. Pat. No. 4,344,438 is of interest for disclosing a fiber-opticchemical sensor that employs a single optical fiber. Here again, a shortsection of dialysis tubing is mounted atop the fiber as ananalyte-permeable indicator-containing housing.

Such fiber-optic probes are small enough to pass through a hypodermicneedle and flexible enough to be threaded through blood vessels forphysiological studies. However, promising medical applications, such ascontinuous blood gas monitoring, have been hindered because experiencehas shown that such probes are difficult and expensive to manufactureand calibrate. Each probe must be exactingly constructed by hand under amicroscope, a process that requires several hours per probe.Considerable unit-to-unit variability in calibration requirementsresults from the slight variations in the assembled configuration of thecomponents. The unique signal response of each hand-crafted probe mustbe calibrated at the time of the assay, typically with at least tworeference pH or other analyte concentration values to adequately definethe calibration curve.

SUMMARY OF THE INVENTION

The invention provides a drift-free fiber-optic sensor suitable formonitoring physiological analyte concentration. An analyte-permeablematrix is disposed in the light path defined by the axial core at oneend of an optical fiber segment. The matrix contains an indicatormolecule covalently linked to a polymer, preferably methylmethacrylate/methacrylamidopropyltrimethylammonium chloride,N-vinylpyrrolidone/p-aminostyrene, methyl methacrylate/hydroxymethylmethacrylate, methyl methacrylate/N-vinylpyrrolidone, or methylmethacrylate/acrylic acid. Such polymers are preferably formulated inthe range of from about 60:40 to about 80:20 wt/wt percent. Inrepresentative embodiments, the polymer is approximately 94:6 mole/molepercent of either methylmethacrylate/methacrylamidopropyltrimethylammonium chloride orN-vinylpyrrolidone/p-aminostyrene copolymer. Drift-free performance isobtained with such sensors having analyte-permeable matrices ofsignificantly less than about 70 microns in thickness.

The indicator molecule, which is capable of responding to a targetedanalyte in an optically detectable manner, is advantageously covalentlylinked to the polymer through an aminoarylalkylamine, such as4-(aminophenyl)-ethylamine or 4-(aminophenyl)-propylamine. The indicatormolecule may be an absorptive molecule, such as phenol red orcarboxynaphthophthalein (hydrogen ion analyte), in which case theindicator molecule may be covalently linked to the polymer througheither an azo-amide or an amidyl-amide linkage. The indicator moleculemay be a luminescent molecule, such as carboxynaphthofluorescein(hydrogen ion analyte) or an oxygen-quenchable porphyrin derivative.

The subject sensor may be provided with a reflector disposed in thelight path distal with respect to the optical fiber segment to theanalyte-permeable matrix. Suitable reflectors include gold foil or filmsof titanium dioxide, zinc oxide, or barium sulfate.

A pCO₂ sensor is configured with a gas-permeable but ion-impermeablemembrane encapsulating an analyte-permeable matrix that includes a basehaving a pKa ranging from about 6.0 to about 7.8. The outer membrane maybe a silicone, polycarbonate, or urethane. The base may be an inorganicsalt, such as bicarbonate, in which case the analyte-permeable matrixshould include an antioxidant. Alternatively, the base may be apolymeric base containing, e.g., 2-vinylpyridine, 4-vinylpyridine,histamine, 1-vinylimidazole, or 4-vinylimadazole. Gas-permeability isafforded to the matrix by a minor component of hydrophilic polymer suchas polyethylene glycol.

A plurality of the foregoing pH, pO₂, and pCO₂ sensors may be disposedtogether in substantially coterminal array to make a multi-variableprobe, which may also include a thermocouple wire. To minimize bloodclotting during in vivo use, the multi-variable probe is preferablyconfigured with the pCO₂ sensor distally disposed to pH and pO₂ sensorsthat are substantially colinearly arrayed.

Also provided is a method of mass producing a fiber-optic sensorsuitable for monitoring physiological analyte concentration. A polymerfilm of substantially uniform thickness is cast, the polymer film beingpermeable to an analyte in solution and including a covalently-linked oradmixed indicator molecule capable of responding to the analyte in anoptically detectable manner. From the film are cut or punched amultiplicity of disc-shaped indicator matrices. The individual indicatormatrices are affixed to optical fiber segments to produce fiber-opticsensors having substantially uniform performance characteristics.

The subject probes are employed to monitor analyte concentration in afluid, by contacting the fluid with the analyte-permeable matrix of thefiber-optic sensor, irradiating the matrix through the optical fibersegment at a first wavelength band corresponding to a region ofanalyte-dependent absorbance by the indicator molecule, measuringabsorbance by or emission from the analyte-permeable matrix at apredetermined second wavelength band, and determining the concentrationof the analyte in the fluid as a function of the measured absorbance oremission. To minimize blood clotting, the sensor should be insertedthrough a catheter means into a patient's bloodstream so that theanalyte-permeable matrix projects from about 0.5 to about 1.5 mm beyondthe end of the catheter means into the bloodstream.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic cross section of a representative pH sensor;

FIG. 2 is a schematic cross section of a representative pO₂ sensor;

FIG. 3 is a schematic cross section of a representative pCO₂ sensor; and

FIG. 4 is a representative multi-variable probe suitable for real-timemonitoring of pH, pO₂, pCO₂, and temperature in the bloodstream.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring to FIG. 1, fiber-optic sensor 10 has an analyte-permeableindicator matrix 12 coated on one end of an optical fiber segment 14.Covalently linked to the indicator matrix 12 is an indicator moleculethat responds to the presence of a specific, targeted analyte in anoptically detectable manner. The indicator matrix 12 is permeable to theanalyte and has a thickness of about 70 microns or less, preferably onthe order of 50 to 15 microns.

By optical fiber 14 is meant a fine, transparent filament, a compositeof two materials having different refractive indices, that is capable oftransmitting electromagnetic radiation. An optical fiber 14, or lightguide, suitable for practicing the invention has an axial transmissivecore that is coaxially surrounded by a cladding material of lowerrefractive index. The cladding serves to confine electromagnetic energyto the core region of the fiber by substantially total reflection at thecore-cladding interface. An optical fiber segment 14 suitable formonitoring physiological homeostasis may have a diameter of about 250 or114 microns and a length on the order of 0.5 meter or more. The opticalfiber segment 14 may be composed of glasses but is preferably made ofplastics.

To manufacture the fiber-optic sensor 10, a clean fiber end is firstprepared, that is, optical fiber segment 14 is cleaved and polished toproduce a square, smooth fiber end. Such a clean flat fiber end can beprepared by procedures well known in the art by jointing optical fibers.The indicator matrix 12 is then applied to the fiber end. In oneembodiment, the fiber end is painted with a resin emulsion containing aresin in a solvent carrier. The resin is selected to render theresulting indicator matrix 12 permeable to the analyte in the testenvironment, and contains a polymer in which an analyte-sensitiveindicator molecule is covalently linked (or, for certain indicatormolecules, admixed). The resin emulsion may be deposited on the end ofthe fiber 14 by dip coating, brushing, spraying, or other conventionalcoating techniques. The solvent carrier is thereafter drawn off, e.g.,by evaporation, to leave an indicator matrix 12 adhering to one end ofthe optical fiber 14. In an alternative embodiment, a disklike indicatormatrix 12 is performed and affixed to the fiber end. As discussed below,a reflective foil 16 may also be provided distal to the indicator matrix12. The resulting fiber-optic sensor 10 can be coupled to conventionalinstrumentation to detect and monitor ambient analyte concentration inliquid or gaseous test environments.

The choice of materials to be used in fabricating the fiber-optic sensor10 is influenced by the need to satisfy simultaneously manyrequirements. Most importantly, the indicator matrix 12 must immobilizethe indicator molecule in the light path defined by the axial core ofthe fiber 14. Otherwise, signal drift will result from leakage ofindicator molecules, especially water-soluble molecules like phenol red,from the remarkably thin indicator matrix 12. Water-soluble indicatormolecules must therefore be covalently bonded to a component of theresin 12. The resulting sensor 10 is drift-free, that is, there is nodetectable leakage or diffusion of indicator molecule from the matrix 12in the environment of use over the time course of the assay.

The indicator matrix 12 must also permit the free passage in and out ofthe analyte substance, that is, matrix 12 must be analyte-permeable. Forphysiological applications in which the analyte is dissolved ordispersed in aqueous solutions, the indicator matrix 12 should behydrophilic as well as porous to the analyte substance. However, thehydrophilicity of the matrix 12 must be regulated to prevent undueswelling, with attendant risk of dissociation from the fiber end, whenthe indicator matrix 12 is immersed in aqueous solutions such as blood,lymph, extracellular fluid, and/or serum. For certain applications, thematrix 12 should be semipermeable as well, having minute openings orpores of a size large enough to permit passage of the targeted analytesubstance but sufficiently small so as to preclude passage of certaindissolved or colloidal substances, e.g., fluorescent blood proteins,that might chemically or physically interfere with the assay.

The indicator matrix 12 must also be optically clear. In addition, therefractive index of the matrix 12 should be reasonably well matched tothat of the optical fiber core, in order to minimize light scatteringeffects such as Fresnel losses.

The constituent resin must be capable of forming and sustaining theindicator matrix 12 on the fiber end. The resin must produce homogeneousresin emulsions or solutions, and should be readily soluble inconventional solvents, particularly solvents having high vaporpressures. Inexpensive, readily available solvents such as thosetypically used for painting and coating applications are preferred. Theresin emulsion or coating solution should have good film-formingproperties, including uniform flow of the solvent casting solution, asphysical anomalities such as bubbles in the matrix 12 can cause signalnoise.

The matrix 12 should not shrink or crack upon drying and should notswell noticeably in aqueous solutions, as there should be nodifferential movement of the indicator matrix 12 vis-a-vis thelight-transmitting fiber core during the time course of use. Theindicator matrix 12 should also retain its rigidity and strength duringuse, e.g., by having sufficient wet mechanical strength to maintain itsintegrity while being manipulated through blood vessels. Sufficient wetadhesion strength between the matrix 12 and the fiber is likewiserequired, and plastic optical fibers 14 are preferred over glasscomposites for having more available bonding sites for film adhesion.Plastic fibers 14 are also relatively inexpensive and easy to cleave,polish, and bend. The high ductility of plastic fibers 14 isadvantageous for in vivo applications. Glass fibers 14 can beconventionally surface treated to increase the adhesion of the indicatormatrix 12, such as by the use of silanes. Such surface-treated glassfibers 14 have transmission advantages for operating at shortwavelengths below the visible. For in vivo blood sensors 10, plasticfibers 14 having polymethyl methacrylate cores have several advantagesover glass fibers, including bendability, thinness, low cost, and easeof cleaving.

The thickness of the indicator matrix 12 over the axial fiber core canvary uniformly in the range of from about five microns to about severalhundred microns, the preferable upper limit being about seventy microns,and most preferably 20 to 35 microns, in order to minimize response timeand light scattering effects. The indicator matrix 12 must uniformlycover at least the light-transmitting core on the end of the fiber 14.In practice, the matrix 12 can in addition overlap the cladding on thefiber end, as well as adjacent lateral surfaces of the fiber 14.

For physiological sensors such as 10, a resin that satisfies theforegoing requirements is made by copolymerizing a mixture of about 94mole percent (mole %) methyl methacrylate (MMA) and about 6 mole %methacrylamidopropyltrimethylammonium chloride (MAPTAC; U.S. Pat. No.4,434,249). Polymethyl methacrylate-based material is an especiallyappropriate matrix component, because of good refractive index match,when used with plastic optical fibers 14 having methyl methacrylatecores. The above-stated copolymer is highly permeable to water and smallions, especially anions, while retaining all the advantages mentionedabove. Methyl methacrylate can alternatively be copolymerized or alloyedwith other ionogenous or neutral monomers, such as hydroxymethylmethacrylate, N-vinylpyrrolidone, or acrylic acid, to confer analytepermeability to the resulting matrix 12.N-vinylpyrrolidone/p-aminostyrene copolymer 60:40 to 80:20 wt/wt % isanother suitable resin material. Suitable solvents for these resins areknown to include alcohols, N,N-dimethylacetamide (DMAC),N,N-dimethylformamide, methyl ethyl ketone, tetrahydrofuran, esters,aromatic and chlorinated hydrocarbons.

The indicator molecule is selected to respond optically in the presenceof the targeted analyte substance when immobilized in the indicatormatrix 12. The response of the indicator molecule should be highlyspecific for the targeted analyte in order to minimize interference andbackground signal noise. For continuous monitoring of analyteconcentration, the reaction or response between the indicator moleculeand the analyte should be reversible as well as sensitive and specific.Suitable analyte-sensitive indicator molecules are known in the art andcan be selected based upon the particular analyte substance whosedetection is targeted and upon other factors as described herein.

Covalent bonding functions to immobilize water-soluble indicatormolecules within the indicator matrix 12 but otherwise must notsignificantly impact upon the sensitivity, specificity, andreversibility of its optical response to the analyte. Thus,analyte-sensitive sites on the indicator molecule must not be eliminatedor sterically hindered upon covalent binding to the resin. The indicatormolecule should therefore be uniformly bound to the resin in asite-specific manner that preserves the optical responsiveness of theindicator molecule to the analyte, using a reaction protocol thatprevents or substantially eliminates heterogeneous reaction products.

For this purpose, aminoarylalkylamines are preferably employed tocovalently link the indicator molecule to a polymer, which is thereafteradmixed in solvent with other resin components to form an emulsion orsolution which may be painted on the fiber end. Suitableaminoarylalkylamines have the formula:

    NH.sub.2 Ar(CH.sub.2).sub.n NH.sub.2

wherein Ar is nonsubstituted or preferably substituted phenyl and n isan integer. Preferably, n equals 2 or 3, in order to avoid hydrocarboncharacteristics associated with longer alkyl chains, which in the caseof pH indicator molecules tend to unacceptably displace the pKa of thelinked indicator. The aminoarylalkylamine is preferablypara-substituted. Exemplary aminoarylalkylamines for practicing theinvention are 4-(aminophenyl)-ethylamine and4-(aminophenyl)-propylamine.

Heterogeneous reaction products are prevented by specifically attachingthe alkylamino moiety to the polymer before reacting the arylaminomoiety with the indicator molecule. The aminoarylalkylamine is firstattached to a polymeric resin component, such as MMA/MAPTAC, by reactionin ethanol at 70° C. with triethylamine as a catalyst. The freearylamino group is then reacted with the indicator molecule of choice,for example by diazotization and coupling with indicator molecules suchas phenol red that bear strongly electron-releasing groups, or byformation of an amidyl linkage with carboxylic acid-bearing indicatormolecules. The available diazonium binding sites should be saturatedwith an excess of indicator molecules during this second reaction step,in order to provide a polymeric resin component containing aconcentrated amount of the indicator molecule. Suitable protocols areset forth below in Examples 1 to 3.

In an exemplary sensor 10, a luminescent indicator molecule iscovalently linked through 4-(aminophenyl)-ethylamine to MMA/MAPTACcopolymer in the indicator matrix 12. For example, the fluorescentindicator molecule carboxynaphthofluorescein is thereby incorporatedinto sensor 10 for monitoring the pH of physiological fluids, thecarboxynaphthofluorescein reacting specifically with hydrogen ion in afluorescent manner at physiological pH. A resin emulsion can be preparedby admixing one part polymeric component saturated withcarboxynaphthofluorescein linked through 4-(aminophenyl)-ethylamine,with about nineteen parts other resin component(s), in a suitablesolvent. Suitable protocols are set forth in Examples 4 and 6. Toconstruct the sensor 10, a clean end of an optical fiber 26 is simplydipped into or painted with the admixed resin emulsion so as to coat thefiber end 24 with the emulsion. The solvent is then allowed toevaporate, leaving adhering on the fiber end a proton-permeable matrix12 containing the linked fluorescent indicator molecule. The thicknessof the indicator membrane need be only from about 25 to about 60 μm,preferably about 40 μm or less, when a luminescent indicator molecule isemployed in sensor 10.

The resulting fiber-optic sensors 10 can be coupled to instrumentationsystems known in the art in order to monitor the pH of physiologicalfluids as functions of luminescent intensity or lifetime. For example,the sensor 10 can be threaded through a hypodermic needle to contact theindicator matrix 12 with a patient's bloodstream. The other, proximalend of the fiber-optic segment 14 is coupled to instrumentationincluding a light source and a photodetector. The light sourceirradiates the indicator matrix 12 through the fiber-optic segment 14with light at a wavelength band corresponding to a region ofanalyte-dependent absorbance by the indicator molecule. Luminescentemission, including lifetime and/or intensity of fluorescence orphosphorescence, as transmitted through segment 14 is measured by thephotodetector. The ambient analyte concentration is determined by knowntechniques as a function of the measured luminescent emission, typicallyby comparison with the signal received from one or more control samplesof known analyte concentration.

In a related embodiment, an absorptive pH indicator dye such as phenolred is linked to N-vinylpyrrolidone/p-aminostyrene copolymer (60:40 to80:20 wt/wt) in indicator matrix 22. Alternatively,carboxynaphthopthalein can serve as the indicator molecule, in whichcase the covalent linkage can be effected by amidyl-rather thanazo-coupling; see Examples 5 and 6. Blood pH can be monitored with theresulting sensors 10 as described above, except that reflectednonabsorbance will typically be monitored. That is, the light reflectedby the matrix 12 will be detected after passing through the fiber 14,and a comparison made of the incident and reflected light intensities ata wavelength band at which the indicator molecule optically responds tothe presence and concentration of the targeted analyte (in this case,hydrogen ion).

Particularly if the indicator molecule responds to the analyte substancein an absorptive manner, e.g., to produce a nonluminescent color change,a reflective foil or film 16 may be applied to the surface of theindicator matrix 12 distal to the fiber core. The reflector 16 acts as amirror to change the direction and increase the directivity of the lightbeam, and in addition provides an appropriate background for analyticalresult detection. The reflector 16 can be a thin metal sheet or can benonmetallic. As an example, gold foil 16 can be adhered to the tackycoating of resin emulsion before the solvent fully evaporates from theindicator matrix 12. A metallic foil 16 can alternatively be applied bycathode sputtering. A reflector film 16 can be made by coating thedistal surface of the indicator matrix 12 with shiny, nontoxic metalliccompounds such as titanium dioxide, zinc oxide, and barium sulfatedispersed in a resin matrix. Surprisingly, the reflector film or foil 16need not be permeable to analyte as long as care is taken not to overlapand cover the lateral margin of the analyte-permeable indicator matrix12. When capped by a nonpermeable foil or film such as gold foil 34, theindicator matrix 12 in sensor 10 can range from about 20 to about 50 μmin thickness. Optimal response times are achieved with such matrices 12having about 35 μm thickness.

Referring to FIG. 2, in a related embodiment, an oxygen-quenchablephosphorescent indicator molecule such as a porphyrin compound isincorporated by admixture into a polymer matrix 12 and applied to theend of an optical fiber 14 to make a sensor 18 suitable for monitoringblood oxygen concentration. The relatively high-molecule-weightporphyrin is insoluble in aqueous solution and so need not be covalentlybonded to the polymer matrix 12. Alternatively, aminoethyl-porphyrinderivatives may be covalently bonded within gas-permeable matrices 12 asdescribed below. The phosphorescent indicator molecule is preferablyselected from among platinum or palladium derivatives oftetrafluorophenylporphyrin, octaethylporphyrin, tetraphenylporphyrin,tetrabenzporphyrin, tetrafluorobenz-porphyrin, andtetrachlorobenzporphyrin. Particularly preferred are photostable,fluorinated derivatives of such metalloporphyrins. In the physiologicaloxygen range of 0-150 torr, platinum tetraphenylporphyrin provides alifetime curve that is especially suitable for determining pO₂. Suchluminescent indicator matrices 12 are preferably from about 70 to about80 μm in thickness with a mirror 16, and from about 120 to about 160 μmin thickness without a mirror 16 (as shown). See Example 8.

FIG. 3 shows an embodiment 20 suitable for monitoring analyte indirectlyas a function of an analyte reaction product. As above, an indicatormatrix 12 is coated on an end of an optical fiber 14, but here theindicator molecule is capable of reacting with an analyte reactionproduct (but not with the analyte per se) in an optically detectablemanner. The indicator matrix 12 additionally incorporates ananalyte-reactive substance that is capable of reacting with the targetedanalyte to produce the analyte reaction product. The indicator matrix 12is overcoated with at least one membrane 22 that is selectivelypermeable to the analyte but not to the analyte reaction product. Thismembrane 22 need not be transparent. A reflector film or foil 16 may beattached to the distal surface of either the indicator matrix 12 or themembrane 20, if the latter is transparent.

In an exemplary pCO₂ sensor 18, the indicator matrix 12 contains phenolred as an indicator molecule codissolved in [MMA/MAPTAC (94:6)-PEG 600K](80:20). A representative protocol is set forth in Example 9. One partof phenol red, sodium salt is admixed with about nineteen parts ofmatrix [(MMA/MAPTAC)/PEG 600K] (80:20). Suitable matrices 12 of thistype can range from about 15 to about 30 μm, preferably about 20 μm, inuniform thickness. The indicator matrix 12 in physiological sensor 18additionally incorporates a base having a pKa ranging from about 6.0 toabout 7.8, and preferably 7.0 to 7.5, which ranges overlap the normalphysiological range of 7.35 to 7.45. A reflective film 16 of titaniumdioxide dispersed in a nontoxic resin matrix is applied to the surfaceof the matrix 12 distal to the fiber core. The reflector 34 and lateralmargins of the matrix 12 are then encapsulated by a gas-permeable bution-impermeable silicone membrane 22 such as SC-32 (General Electric). Asuitable encapsulating membrane 22 can alternatively be formed fromcommercially available polycarbonate or urethane resins. Membrane 22 canhave a thickness on the order of about 50 microns. A second membrane 24may be added to envelop this first membrane 22, in order to affordstructural stability to the probe 18. For example, a resilient outermembrane 24 of SC-35 silicone (Huls America) can be employed inconjunction with an inner membrane 22 of SC-32 silicone, the latterserving to sequester the alkaline indicator matrix 12 from thepH-sensitive SC-35 silicone. In practice, the [(MMA/MAPTAC)/PEG 600K]component of matrix 12 must be formulated to afford hydrophilicity butwithout undue swelling that would breach the outer silicone membrane(s)22, 24. Sufficient hydrophilicity is afforded by the 80:20 formulationdescribed above, while undue swelling tends to occur when the copolymeris formulated below 60:40 wt/wt %.

The base that is incorporated into indicator matrix 12 can be aninorganic salt such as a bicarbonate, preferably sodium bicarbonate. Thebicarbonate can be incorporated into sensor 20 by soaking the indicatormatrix-tipped fiber end in aqueous bicarbonate solution (18 hours in a35 mmol concentration will suffice), either prior or subsequent toapplying reflector 16 to the matrix 12, but before membrane 22 isapplied. Such a sensor 20 must additionally incorporate an antioxidantcompound, or sterilization of this particular embodiment 20 with E-beam(2.5 Mrads) results in drastic degradation of the system, with apparentloss of sodium bicarbonate, resulting in unacceptable performance. Asuitable antioxidant for this purpose is1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-hydroxybenzyl) benzene(ETANOX 330; Ethyl Corporation, Baton Rouge, La.). The antioxidant canbe admixed in the bicarbonate soaking solution at concentrations on theorder of 0.1% to 0.2%.

The above inorganic base can advantageously be replaced by a polymericbase having a pKa in the targeted range. Suitable monomeric bases forphysiological studies include but are not limited to 2-vinylpyridine,4-vinylpyridine, histamine, 1-vinylimidazole, and 4-vinylimidazole. Thevinylic monomers can be homopolymerized or copolymerized to provide apolymeric base of sufficiently high molecular weight to nullify loss bypermeation, when physically entrained in matrix 12, during or afterE-beam sterilization. Any of the above monomers can alternatively becopolymerized with MMA or otherwise bonded to the resin emulsion, inorder to immobilize the base by covalent linkage to the indicator matrix12. A preferred polymeric base for the above purpose is the basic formof the PAMM dye. See Example 10.

Whether an inorganic or polymeric base is incorporated into pCO₂ sensor20, the indicator matrix 12 must be sufficiently hydrophilic to permitan equilibrating entry and egress of carbon dioxide into matrix 12 atrates conducive to acceptable response times for clinical uses. An 80:20wt/wt percent [(MMA/MAPTAC)/PEG 600K] matrix 12 is sufficientlyhydrophilic to make real-time monitoring of blood pCO₂ feasible. Otherresins suitable for imparting hydrophilicity to the matrix 12, bycopolymerization or alloying with other resin components, includepoly-N-vinylpyrolidone, polyethylene oxide, methocel, and polyurethane.

Sensor 20 can be inserted into the bloodstream to monitor pCO₂.Indicator matrix 12 is then irradiated in situ through fiber 14 withlight at a wavelength band corresponding to a region of proton-sensitiveabsorbance by the covalently linked indicator molecule. Reflected lightat that same wavelength band, as returned through fiber 14, is typicallymeasured. The ambient carbon dioxide concentration is typicallydetermined as a function of reflected nonabsorbance, by comparing theingoing and outgoing intensities with reference to one or more samplesof known carbon dioxide concentration.

The disclosed fiber-optic chemical sensors 10, 18, 20 are manufacturedusing exclusively solvent-based film coating techniques, with ananalyte-sensitive indicator molecule covalently attached to or admixedin the matrix formulation 12. This process lends itself directly to massproduction of the fiber-optic chemical sensors, particularly by therepresentative procedures disclosed in Example 11. The sensors can besimultaneously and uniformly manufactured by dipping, spraying, orotherwise painting the indicator matrix 12 onto the registered ends ofbundled fibers 14, or uniformly preformed indicator matrices 12 (with orwithout reflectors 16) can be applied to individual fibers 14 at arobotized workstation. A gold mirror or other reflective metallic layer16 may then be applied, followed by an overcoat of a semipermeablemembrane 22 if desired. An overcoating film of a blood-clot-inhibitingsubstance may then be applied; see Example 10. The resulting sensors areindividually packaged in sterile containers, such as plastic bags,typically accompanied by printed instructions for their use.

Sterility of the sensors 10, 18, 20 may be conferred by knownirradiation, e.g., E-beam or chemical e.g., ethylene oxide (EtO),treatments. E-beam sterilization is preferable for avoiding residual EtOpresence. The sensors can be packaged in a hydrated condition for E-beamsterilization, which makes for more convenient packaging, handling, andcalibration. Furthermore, dehydration of the sensors, which is requiredduring EtO sterilization, may impair sensor function, especially the pHsensor 10. However, the pCO₂ probe 20 containing nonpolymerizedinorganic base in matrix 12 is not susceptible to E-beam sterilization.Provision of a polymeric base in matrix 12 renders sensor 20advantageously susceptible to E-beam sterilization.

Referring to FIG. 4, a multi-variable probe includes the pH sensor 12,the pO₂ sensor 18, the pCO₂ sensor 20, and/or a thermocouple wire 30arranged in fixed array. The thermocouple wire 30 may be employed tomeasure temperature at the probe site, which temperature information maybe used to correct any temperature sensitivity of the other sensors, orfor compensation of the measured blood-gas parameters to that whichwould be read by a blood-gas analyzer at 37° C. This multi-variableprobe 26 is preferably configured to protrude through a conventional20-gage catheter into a patient's radial artery. A preferred protocolfor constructing probe 26 is set forth in Example 13.

The subject probes 10, 18, 20, 26 are preferably coated with ananticoagulant film 34, as discussed in Example 12.

Prior to use the probes must be hydrated and calibrated. The nature ofthe matrices 12 requires that a hydration sequence must be followed toachieve the desired performance. For example, MMA/MAPTAC is a materialwhose majority component is hydrophobic but whose minority component(component allowing gas and ion transfer) is hydrophilic. Thus, it isnecessary to hydrate both the pH and pCO₂ systems 10, 20 a minimum ofone week at an elevated temperature (40° C.) to increase the hydrophilicnature of the matrix 12. The hydration fluid can be a phosphate bufferedsystem (pH 7.0 to 7.4).

The subject O₂ system 18 need not be hydrated and may be directlycalibrated with atmospheric oxygen.

Following hydration, calibration of the probe 10, 20 can be achieved byplacing the probe in a phosphate buffer 6.8 to 7.1 and allowing this toequilibrate. Upon equilibration the probe can be transfered into anotherbuffer 7.4 to 7.8. Each buffer should also contain a known pCO₂ and pO₂content, comparable with the normal blood concentrations of interest, topermit calibration of each parameter.

In use, the positioning of the sensor 10, 18, 20, 26 with respect to thecatheter through which it is threaded is very important to minimizeblood clotting. If the position is too far advanced, the probe will clotmore frequently than desirable. If the position is not advanced farenough, e.g., positioned within the catheter (negatively extended), theprobe will not accurately measure the blood parameters, or thethermocouple measurement may not accurately measure the bloodtemperature. Thus, the tip of probe 26 should be positioned from about0.5 to about 1.5 mm extended past the catheter tip. This narrow range of1.0 mm must be maintained to obtain the desired sensor performance whileminimizing blood clotting.

The following Examples are provided to illustrate the advantages and toassist one of ordinary skill in making and using the invention. TheExamples are not intended in any way to otherwise limit the scope of thedisclosure and the protection granted by Letters Patent hereon.

EXAMPLE 1 Preparation of MMA/MAPTAC Polymer

Methyl methacrylate (MMA) and methacrylamidopropyltrimethylammoniumchloride (MAPTAC) were obtained from Polysciences, Warrington, Pa.

A mixture of 1.7 ml of 50 wt % aqueous MAPTAC (purified by charcoalchromatography, 0.0040 moles MAPTAC, 0.05 moles water), 6.7 ml ofdistilled MMA (0.06 moles), 4 ml of ethanol, and 20 mg of2,2'-azobisisobutyronitrile (AIBN; Polysciences) was stored in a sealedvial at 75° C. for 24 hours. The resulting 94:6 mol/mol percent polymerwas removed, and a solution of the polymer in denatured ethanol wasprepared by stirring 5 g of the polymer in 50 of denatured alcoholovernight.

EXAMPLE 2 Attachment of APE to MMA/MAPTAC Polymer

4-(aminophenyl)-ethylamine (APE) was purified as the dihydrochloride bytaking 4 g of APE (Aldrich Chemical Co., Inc., Milwaukee, Wis.) in 8 mlof concentrated hydrochloric acid at 0° C. and recrystallizing thedihydrochloride from water-ethanol (100 ml of 95:5 water-ethanol).

2 ml of the 10% MMA/MAPTAC solution from Example 1 was azeotroped withanhydrous ethanol (3×50 ml) and redissolved in 25 ml anhydrous ethanol0.38 g of the APE-dihydrochloride and 1 ml of freshly distilledtriethylamine (Aldrich) as a catalyst were added, and the solutionstirred in an oven at 55° C. for 48 hours. The solvent and excesstriethylamine were removed on a rotary evaporator.

EXAMPLE 3 Diazotization and Coupling of Phenol Red to APE/MMA/MAPTAC

The APE/MMA/MAPTAC reaction product from Example 2 was dissolved in 20ml of denatured ethanol at 0° C., and to that solution was added 3 ml ofconcentrated HCl and 3 ml of water. A solution of 0.3 g of NaNO₂ in 2 mlof water was then added, and the solution stirred at 0° C. for threehours. This was then added to a solution of 2.4 g phenol red (Na-salt ofphenol red; Aldrich) and 2.5 g of KHCO₃ in 30 ml of water and 30 ml ofdenatured ethyl alcohol, while stirring at 0° C. It is important, whencoupling the diazotized APE polymer to phenol red, to maintain pH atabout 8.5 using KHCO₃, and to use excess phenol red to saturate alldiazotized sites and prevent diazonium hydroxide/phenol formation. Theresulting solution was stirred overnight at 0° C.

The resulting orange-red solution from the coupling reaction was broughtto pH=1 with concentrated HCl at 0° C., and 500 ml of ice-cold wateradded. The product was filtered and the residue washed with water (3×100ml).

The crude product was mixed with 2.5 g of KHCO₃ in 250 ml water, and astirred cell separation was conducted using a type F membrane (SpectrumUltrapor,

Type F MWCO: 50,000: Spectrum Medical Industries, Los Angeles, Calif.)under nitrogen gas. The ultrafiltration was continued until the filtratewas colorless, as indicated by nonabsorption at 570 nm. Thereddish-brown pure product was dried in a dessicator. A 15% (wt/wt)solution of this product on MMA/MAPTAC polymer solids met allspecifications for a pH sensor.

EXAMPLE 4 Preparation of Carboxynaphthofluorescein (CNF)

A mixture of 2 g of trimelletic anhydride and 3.2 g of1,3-dihydroxynaphthalene (Aldrich Chemical Co.) was heated to 200° C.,and 5 g of freshly fused zinc chloride was added in portions. The moltenmixture was heated at 200° C. for 1.5 hours, cooled to 100° C., and(2×25) ml of 50° HCl was added. The intensely fluorescent solution wassaturated with NaCl, filtered, and the residue washed with water to givea reddish-brown solid which was recrystallized from ethanol.

EXAMPLE 5 Preparation of Carboxynaphthophthalein (CNP)

A mixture of 19 g of trimelletic anhydride (Aldrich Chemical Co.) and 28g of α-naphthol (Aldrich Chemical Co.) was heated with 11 ml ofconcentrated sulfuric acid at 120° C. for 8 hours. The reaction mixtureturned deep green and was extracted with boiling water (2 liters). Theaqueous extract was filtered and the residue washed with water. Thecrude product was then dissolved in sodium hydroxide (pH 10.5) and thesolution filtered. The green filtrate was acidified to pH 4 with glacialacetic acid, and the brown residue filtered and recrystallized fromethanol.

EXAMPLE 6 Attachment of CNF (or CNP) to APE/MMA/MAPTAC

To a stirred, cold solution of 2.5 g of the above CNF (or CNP) in 50 mlof dichloromethane was slowly added 0.60 g of dicyclohexylcarbodimide.The solution was stirred for 15 minutes. A solution of APE/MMA/MAPTAC(from Example 2, in 25 ml dichloromethane) was added, and the resultingsolution was stirred overnight. The precipitated dicyclohexyl urea wasfiltered, and the solvent was removed on a rotary evaporator. Theresidue was then purified by ultrafiltration (MWCO: 50,000) and used forprobe construction.

EXAMPLE 7 Preparation of DEF-1 (pH Dye)

Weigh out 3.07 g of a 13% (wt/wt) solution of MMA/MAPTAC (94:6) in2-methoxyethanol (0.4 g solid MMA/MAPTAC). Add 0.06 g PAMM(PR:APE:MMA:MAPTAC) 15% wt/wt on solid MMA/MAPTAC. Solution is stirreduntil homogeneous.

EXAMPLE 8 Preparation of PT55 (PtTFPP and SC-35) Film (pO₂ Dye)

0.25 g of SC-35 silicone (Huls America) and 0.012 g PtTFPP (PorphyrinProducts, Logan, Utah) were weighed and mixed together. The percentageof material in solution can be adjusted, depending upon how sensor 18 isto be constructed. For hand-building individual sensors 18, 9.74 g oftetrahydrofuran (THF) can be added to the above mixture, to provide asolution in which individual fiber ends can be dipped to form matrix 12.For mass production techniques, a 10% solution can be made by adding2.36 g of THF to the above constituents.

EXAMPLE 9 Preparation of DYE54C Film (pCO₂)

To prepare a solution of PEG 600K, dissolve 1.0 g solid PEG 600K in 19 gof 2-methoxyethanol (5% wt/wt) and stir or sonicate until homogeneous.To prepare a solution of MMA/MAPTAC (94:6), dissolve 1.0 g solidMMA/MAPTAC in 6.7 g of 2-methoxyethanol (13% wt/wt) and stir untilhomogeneous. Mix 3.07 g of the 13% MMA/MAPTAC solution with 2.0 g of the5.0% PEG 600K solution. The ratio of solid MMA/MAPTAC to solid PEG 600Kis 80% to 20%. Sonicate until admixed solution is homogeneous. To themixed solution add 0.005 g of phenol red and stir until homogeneous. Add200 μl of the 0.875 Molar bicarbonate solution (sodium bicarbonate 0.875Molar).

EXAMPLE 10 Preparation of CAP-2 Film (pCO₂)

Weigh out 3.07 g of a 13% (wt/wt) solution MMA/MAPTAC (94:6) in DMAC.Add 2.0 g of a 5% (wt/wt) solution of PEG 600K (polyethylene oxide 600K)in DMAC. Add 0.075 g of 15% PAMM (PR:APE:MMA:MAPTAC) dye in its basicred form. The solution is stirred until homogeneous. The % total isbrought to 9% wt/wt by adding 1.40 g DMAC.

EXAMPLE 11 Mass Production Protocols

Pelletization of the materials is preferably used in fabricating thefiber optic sensors and affords several advantages. In the dipped orpainted version of the fiber optic sensor, dye concentration isachievable but matrix-length control is harder to achieve for ahigh-volume manufacturing process. The pelletization approach allows auniform film thickness (i.e., matrix 12 length) with a mirror support,if desirable. The manufacturing process involves placing this dye-coatedmirror (i.e., pellet) on the fiber tip and encapsulating it with amembrane if required. The concentration x length control that can beachieved in this manner gives a highly reliable and accurate probe withthe potential for a one-point calibration.

Preparation and mounting of gold foil for pCO₂ and pO₂ films: Gold foilis received in 1"×12" strips (2.5 cm by 30 cm) from Cominco ElectronicsMaterials of Spokane, Wash., preferably shipped in a plastic rollinstead of a flat pack. Begin preparing the gold foil for pCO₂ or pO₂films by placing the foil between two clean glass slides, and cuttingaway a 1 cm by 2.5 cm strip. Cut the strip in half again, such thatthere are two 1 cm by 1.25 cm pieces. Measure each piece of foil forthickness using, e.g., a Mitutoyo Digital Micrometer; check foruniformity. Place each piece of foil in a scintillation vial and addabout 1 ml of concentrated HCl. Allow the foil to soak in theconcentrated HCl for at least 2 hours (preferably overnight) to removeany residues on the foil surface. Remove the piece of gold foil from thevial of concentrated HCl with bull-nosed tweezers, and rinse each sideof the foil with copious amounts of distilled H₂ O, three times for eachside. Place the gold foil on a glass slide, and remove any moisture fromthe gold surface with blotting paper. Examine both sides of the foil forshininess/impurities. If spots/impurities do appear on the foil, placeit back into concentrated HCl and begin again.

With the gold foil on a glass slide, use adhesive tape to anchor twoopposite sides of the gold foil to the glass surface. Tape the gold foildown such that the surface of the foil is flat (you can stretch the foilflat somewhat after it has been taped down), and a 1 cm by 1 cm area ofthe foil is exposed. Next, mask the tape to prevent the dye solventsfrom dissolving the tape mount and, hence, destroying the film prep.Place a bead of UV-curable adhesive (e.g., NOA-81; Norland Products,Inc., New Brunswick, N.J.) along the tape on both sides of the goldfoil. Using a #2 paintbrush, bring the adhesive over the tape, and rightup to the surface of the foil, but do not get adhesive on the surface ofthe gold foil. Should the NOA-81 adhesive leach onto the gold surface,simply cure the adhesive under a 365 nm UV lamp, peel the cured adhesiveaway, and begin applying the NOA-81 again. Once the NOA-81 has beenbrought to the edge of the foil (on both sides), such that it completelycovers the tape but is not over the gold surface, cure the NOA-81 byplacing it under a 365 nm UV lamp. Allow the adhesive to cure for about10 minutes.

Preparation of CAP-2 films (pCO₂ dye)

1) Prepare a 9 wt % solution of CAP-2 in DMAC, as described in Example8.

2) Mount a 1 square cm piece of gold foil as described above.

3) Place a leveling plate on top of a Corning Hotplate/Stirrer and setthe heat to LOW (45° C.).

4) Using a two-way level, adjust the height of the screws on theleveling plate until the plate is level.

5) Place the glass slide containing the gold foil mount onto theleveling plate; allow the setup to achieve temperature equilibrium.

6) Place the 9% solution of CAP-2 into the oven, and allow it to reach45° C.

7) Place a 50 μl aliquot of the 9% CAP-2 solution onto the surface ofthe gold foil with a micropipette. Use the micropipette tip to brush thedye over the entire surface of the gold. Be careful not to brush the dyebeyond the foil edge. Should this happen, remove the sample and beginagain with a new foil mount. Remove any bubbles in the film surface byblowing air through the micropipette tip.

The measured amounts of dye given for the film preps, here and below,are based on an exposed gold foil area of 1 cm² (i.e., 50 μl/cm²). Formounted foils having exposed surface areas other than 1 cm², simplymultiply the exposed area by the amount of dye given for 1 cm², andapply that amount of dye to the foil surface.

8) Place a 7 cm drying tube over the sample. Be sure not to move theleveling plate, or touch the film surface. Allow the film to dry. Thisshould take about 2 hours.

9) Observe the quality of the dry film surface. Eliminate film samplesthat are not smooth or that exhibit impurities in the film.

10) Use a sharp razor blade to cut the sample from its mount. Place thesample under the Mitutoyo Digital Micrometer and measure its thickness.Make several measurements covering the entire surface of the film.Record the range of thickness measurement (for example, 36-42 μm), aswell as the most common thickness measured. Film thickness should notvary by more than 8 μm in the area of the film which is to be punchedfor indicator matrices 12. The film may now be mounted for punching.

Preparation of PT55 films (pO₂ dye)

1) Prepare a 10 wt % solution of PT55 in THF, as described in Example 7.

2) Mount a 1 square cm piece of gold foil as described above.

3) Place the glass slide containing the gold foil mount onto a leveledheating plate at room temperature.

4) Place a 100 μl aliquot of the 10% PT55 solution onto the surface ofthe gold foil with a micropipette. Use the micropipette tip to brush thedye over the entire surface of the gold. Be careful not to brush the dyebeyond the foil edge. Should this happen, remove the sample and beginagain with a new foil mount.

When applying the PT55 dye in THF, it is important to be sure that theedges of the foil are covered with dye first, prior to releasing all ofthe dye from the micropipette tip. Because the film sets up so quickly,once all the dye has been placed on the film, any attempt to move moredye closer to the film edge results in a sticky mess. Equally importantis taking care not to cause bubbles to form in the film during dyedelivery. Bubbles are most often formed when the dye is released tooquickly from the pipette tip. It is a good idea to leave a small amountof dye in the pipette tip, rather than trying to push out every μl.

If delivery of the dye in THF is too difficult, the film can be preppedusing PT55 in Toluene (10 wt %). Dry over low heat (45° C.).

5) Place a 7 cm drying tube over the sample and allow the film to cureovernight. Allowing the film to cure overnight results in an increase ofdye/mirror adhesion.

6) Check the quality of the film surface for smoothness and impuritiessuch as bubbles.

7) Use a sharp razor blade to cut the sample from its mount. Place thesample under the Mitutoyo Digital Micrometer and measure its thickness.Make several measurements covering the entire surface of the film.Record the range of thickness measurement (example 75-85 μm), as well asthe most common thickness measured. Film thickness tends to vary agreater magnitude for the PT55 than the CAP-2 films. However,consistency of film thickness is less significant for the pO₂ system,and a variation of ±7 μm is acceptable. The film may now be mounted forpunching.

Preparation of gold foil for pH films and production mirrors: Thepreparation of gold foil for the making of pH films, and the productionof plain gold mirrors is identical to that given above for pCO₂ and pO₂films, with the exception of the length that the gold foil strip is cut.For pH films and plain gold mirrors, place the gold foil between twoclean glass slides and cut a 2.5 cm by 2.5 cm strip of foil. Cut thestrip in half again, such that there are two 1.25 cm by 1.25 cm pieces.From here, follow the preparation given above for pCO₂ and pO₂ (i.e.,conc. HCl soak, etc.).

Mounting of the gold foil for pH film preparation/application of NOA-81adhesive to the foil surface: Place the gold foil on a clean glassslide. Use adhesive tape to anchor two opposite sides of the gold foilto the glass slide. Tape the gold foil down such that the surface of thefoil is flat (you can stretch the foil flat somewhat after it has beentaped down) and the distance between the two pieces of tape is 1 squarecm. Cut along the edges of the foil which are not taped and remove theexcess adhesive tape with a razor blade. Place adhesive tape over theother two sides of the foil, such that the total exposed surface area ofthe gold foil is 1 square cm and the final two pieces of tape extendover the first two pieces of tape (which were trimmed off right at thefoil edge). Use bull-nosed tweezers to compress the edges of theadhesive tape down on the gold foil. Remove any air pockets between thepieces of adhesive tape and the glass slide and foil. Place a drop ofNOA-81 adhesive on the tape away from the foil surface. Dip a cleanpaint brush into the center of the NOA-81 bead. Using rapid brushstrokes, brush the paint brush tip containing adhesive over the surfaceof the gold foil (about 25 strokes). Rotate the foil 90° and, againusing rapid brush strokes, brush the adhesive in the other direction.Check to see that the adhesive appears to have completely covered thegold surface and that there are no impurities in the adhesive (brushhairs, lumps of adhesive, etc.). Continue rotating and brushing theadhesive until coverage is complete.

To form borders around the foil-backed area that will receive the dye,place a bead of NOA-81 adhesive along the tape on two sides of the goldfoil. Using a #2 paintbrush, bring the adhesive over the tape and rightup to the surface of the foil. Unlike pCO₂ and pO₂ films, should theadhesive leach over the tape onto the gold foil, it cannot be cured andremoved, since doing so would also remove the brushed-on adhesive. Oncethe NOA-81 has been brought to the edge of the foil (on both sides),such that it completely covers the tape, but is not over the goldsurface, cure the NOA-81 by placing it under a 365 nm UV lamp. Allow theadhesive to cure for a few seconds. Place a bead of NOA-81 adhesivealong the tape on the remaining two sides of the gold foil. Using a #2paintbrush, again bring the adhesive over the tape and right up to thesurface of the foil. Again, get adhesive on the surface of the goldfoil. Once the NOA- 81 has been brought to the edge of the foil (on bothsides), such that it completely covers the tape, but is not over thegold surface, cure the NOA-81 by placing it under a 365 nm UV lamp.Allow the adhesive to cure for about five minutes.

Preparation of DEF-1/NOA-81 films (pH Dye)

1) Prepare a 5 wt % solution of DEF-1 in ethoxyethanol. Weigh cut andmix together 0.4 g MMA/MAPTAC solid (acid form) and 0.06 g PAMM(PR:APE:MMA:MAPTAC). Add 8.74 g ethoxymethanol bringing the solids to5%.

2) Prepare a 9 wt % solution of DEF-1 in ethoxyethanol, which contains 5wt % ethylene glycol (on total solids). 0.4 g MMA/MAPTAC, 0.06 g PAMM,4.65 g ethoxyethanol, bringing solids to 9% with 0.023 g ethyleneglycol.

3) Mount a 1 cm² piece of gold foil to a clean glass side and applyNOA-81 as described above.

4) Place the glass slide containing the gold foil mount onto a leveledheating plate to room temperature.

5) Using a spray atomizer, spray the surface of the gold foil with the5% solution of DEF-1/ethoxyethanol such that the gold foil surface iscompletely wetted.

6) Immediately apply 150 μl of 9% DEF-1/ethoxyethanol over the wettedgold foil surface with a digital micropipette (in three 50 μl aliquots).Check to ensure that the dye has not run out of its NOA-81 boundary.

7) Place a 7 cm drying tube over the sample. Place a piece of weighingpaper with a small hole cut in the center over the top of the dryingtube. Allow the film to set up. This should take about 16 to 20 hours,but needs to be monitored closely.

8) The film edges should be checked periodically with a 90° dentalprobe. Once the edges have "set up", set up meaning soft but notcompletely dry, the film must be cut from the glass slide, mounted forpunching, and punched immediately. Failure to punch the film before itis completely dry will result in a high % of cracking and shattering ofthe dye, resulting in loss of adhesion. Thickness measurements for thesepH films are made after punching is complete.

Mounting films/foil for punching: Place the sample which has been cutfrom the glass slide and measured for thickness (pCO₂ and pO₂ filmsonly) on the counter, film side up. Using adhesive tape, tape all foursides of the film to the counter, allowing the tape to cover about 1 mmof the film on each side. Use the end of a bull-nosed tweezers to securethe adhesive tape to the film by compressing the tape down onto the filmsurface, being careful not to scrape the film surface. Trim away anyexcess tape, so that your film mount is square. Remove the film mountfrom the counter and invert it onto a glass slide. Place thin strips ofadhesive tape around the underside of the film, such that the tapeextends over the gold surface but not beyond the tape on the film sideof the sample. Again, use the end of the bull-nosed tweezers to compressthe tape securely against the foil. Center the film mount on themicropunch XY plate, dye side up, and tape it to the XY plate such thatthe film lies flat and there are no folds in the adhesive tape. Checkthe underside of the sample to be sure that the gold foil is clean.Secure the XY plate to the micropunch (e.g., Model #001, Abbott ResearchInc., Bothell, Wash.).

For the production of plain gold mirrors, the mounting procedure is thesame as is given above. The exception, of course, is that there is no"dye side up". In an alternative embodiment, the polymer film of uniformthickness may be cast, and then a reflective surface applied to oneside, e.g., by gold sputtering, prior to pelletization with amicropunch.

During mounting, the pH film is not completely dry. Be careful not tocompress the pH film against the glass slide when placing thin strips oftape on the underside of the foil.

After the film (pH, pCO₂, or pO₂) is mounted, pellets of uniform sizeare punched and collected per the micropunch procedures manual. Thepellets are visually inspected for uniformity.

pH films of DEF-1/NOA-81 should measure between 45-55 μm, preferably46-54 μm, total film thickness.

pCO₂ films of CAP-2 should measure between 30-40 μm total filmthickness. A typical film prepared as outlined previously should measure34-39 μm and have excellent surface quality.

pO₂ films will show the largest thickness variation. Films prepared asoutlined previously should measure to 75±10 μm total film thickness. Aslong as the pO₂ dye thickness is >50 μm, such pellets can be used forsensor building.

EXAMPLE 12 Anti-Clotting Coatings

A polyHEMA film may be applied to any of the foregoing probes, in orderto minimize and retard blood clotting upon the probe during in vivo use.In a representative synthesis, 10 g of HEMA monomer(2-(hydroxyethyl)methacrylate; Polysciences) is passed over Dehibit 100(Polysciences). Weigh out 5.0 g of monomer, add 95 g anhydrous EtOH, add30 mg AIBN, and heat at 60° C. for 48 hrs. Remove the solvent. The whitesolid can be solubilized with methanol, at 2% solids, to form asolvent-castable solution into which the probe 10, 18, 20, 26 is dipped.

Alternative coatings include hydrogels such as pHEMA, HEMA/N-vinylpyrrolidone copolymer, ethylmethacrylate/polyethlene copolymer, andvarious polyurethanes. Such hydrogels are thought to exhibit low proteinsurface adsorption, and are hydrophilic so they allow passage of ionsand gas without changing the sensor response.

EXAMPLE 13 Construction of Multi-Variable Probes

Three 114 μm core optical fibers are cleaved to perpendicular. The fiberends designated to be pH and CO₂ sensors 10, 12 receive two thin coatsof PMMA (not shown). pH dye is dabbed onto the tip of the pH fiber untilthere is a 35 μm layer of dye 12. Then a gold mirror 16 is appliedperpendicular to the fiber axis. Then more dye is applied until themirror is overcoated and its width is between 165 to 175 μm at themirror. Alternatively, a preformed pellet containing the pH dye andmirror backing is affixed, e.g., using a THF solvent, to the end of thefiber so as to orientate the reflective surface perpendicular to thefiber axis.

The CO₂ dye 12' may be painted on the designated fiber end to provide a20 μm layer of dye; or, a preformed pellet may be prepared and appliedas described above. Here, the width of the overcoat should be between135 to 145 μm. Then an overcoat 22 of SC-32 is applied such that thesensor width is between 195 to 205 μm and the overcoat 22 wicks lessthan 150 μm down the fiber from the matrix 12'. After the silicone iscured, it receives an overcoat 24 of SC-35 such that the width isbetween 275 to 300 μm and the wicking is between 300 to 500 μm.

The O₂ sensor 18 receives successive dabs of O₂ dye until a cylinder ofdye 12" between 100 to 140 μm results and wicking at 100 to 200 μm downthe fiber is present. The dye width should be 140 to 160 μm. Here again,the matrix 12" may be advantageously preformed as described above.

Finally, the tips 10, 18, 20 are brought into close proximity using anepoxy 28. For minimizing blood clotting, it has been found to beimportant to stagger the probe components, with the pCO₂ sensor 20arrayed distal to the pH and pO₂ sensors 10, 18, as shown in FIG. 4,rather than to arrange the sensors 10, 18, 20 in colinear array. Inaddition, the sensors should be aggregated with minimal lateral splay,to reduce the overall probe tip diameter and the opportunity for clotthrombogenesis.

A thermocouple wire 30 may be embedded in epoxy 28 near the probe tip,if blood temperature readings are desired. A polyimide sheath 32 may besupplied, distal to the probe tip, to envelop the optical fibers andwire, to provide stiffness and dielectric strength to the probe 26. Theresulting composite probe 26 is typically coated with an anticoagulentmaterial 32.

While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill after reading the foregoingspecification will be able to effect various changes, substitutions ofequivalents, and alterations to the methods and compositions set forthherein. It is therefore intended that the protection granted by LettersPatent hereon be limited only by the definitions contained in theappended claims and equivalents thereof.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A fiber optic sensorsuitable for monitoring physiological analyte concentration, comprisinga preformed film of an analyte permeable matrix disposed in a light pathdefined by an axial core at one end of an optical fiber segment, theanalyte permeable matrix comprising an indicator molecule covalentlylinked to or admixed with a polymer, the indicator molecule capable ofresponding to the analyte in an optically-detectable manner.
 2. Thefiber-optic sensor of claim 1, wherein the polymer is selected from thegroup consisting of methyl methacrylate/methacrylamidopropyltrimethylammonium chloride, N-vinylpyrrolidone/p-aminostyrene, methylmethacrylate/hydroxymethyl methacrylate, methylmethacrylate/N-vinylpyrrolidone, and methyl methacrylate/acrylic acid.3. The fiber-optic sensor of claim 1, wherein the pre-formed film issupported on a reflective film.
 4. The fiber-optic sensor of claim 3,wherein the preformed film is a preformed pellet.
 5. The fiber-opticsensor of claim 2, wherein the polymers formulated in the range are fromabout 60:40 to about 80:20 weight/weight percent.
 6. The fiber-opticsensor of claim 1, wherein the polymer comprises approximately 94:6mole/mole percent of either methylmethacrylate/methylacrylamidopropyltrimethylammonium chloride orN-vinylpyrrolidone/p-aminostyrene copolymer.
 7. The fiber-optic sensorof claim 1, wherein the indicator molecule is covalently linked to thepolymer through an aminoarylalkylamine.
 8. The fiber-optic sensor ofclaim 7, wherein the aminoarylalkylamine is selected from the groupconsisting of 4-(aminophenyl)-ethylamine and4-(aminophenyl)-propylamine.
 9. The fiber-optic sensor of claim 1,wherein the indicator molecule is an absorptive molecule.
 10. Thefiber-optic sensor of claim 9, wherein the analyte is hydrogen ion. 11.The fiber-optic sensor of claim 10, wherein the indicator molecule isselected from the group consisting of phenol red andcarboxynaphthophthalein.
 12. The fiber-optic sensor of claim 1, whereinthe indicator molecule is a luminescent molecule.
 13. The fiber-opticsensor of claim 12, wherein the analyte is hydrogen ion.
 14. Thefiber-optic sensor of claim 12, wherein the analyte is oxygen.
 15. Thefiber-optic sensor of claim 1, further comprising a gas permeable butionimpermeable membrane encapsulating the analyte-permeable matrix, theanalyte-permeable matrix comprising a base having a pKa ranging fromabout 6.0 to about 7.8.
 16. The fiber-optic sensor of claim 15, whereinthe base has a pKa ranging from about 7.0 to 7.4.
 17. The fiber-opticsensor of claim 15, wherein the gas-permeable but ion-impermeablemembrane comprises a resin selected from the group consisting ofsilicones polycarbonates, and urethanes.
 18. The fiber-optic sensor ofclaim 15, wherein the base comprises an inorganic salt and wherein theanalyte-permeable matrix further comprises an antioxidant.
 19. Thefiber-optic sensor of claim 15, wherein the analyte-permeable matrixcomprises one or more of the group consisting ofpoly-N-vinylpyrrolidone, polyethylene glycol, and polyethylene oxide.20. The fiber-optic sensor of claim 15, wherein the analyte-permeablematrix comprises approximately 80:20 weight/weight percent.